Rapid Multi-Locus Sequence Typing Using Microfluidic Biochips

• Type of Material: Article
• Authors: Timothy D Read, Emory University; Rosemary S. Turingan, Emory University; Christopher Cook, Emory University; Heidi Giese, Network Biosystems Inc.; Hans Thomann, Network Biosystems Inc.; Catherine C. Hogan, Network Biosystems Inc.; Eugene Tan, Network Biosystems Inc.; Richard F. Selden, Network Biosystems Inc.
• Language: English
• Date: 2010
• Publisher: Public Library of Science
• Publication Version: Final Published Version
• Copyright Statement: Copyright Read et al.
• Final Published Version (URL): http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0010595
• Title of Journal or Parent Work: PLoS ONE
• ISSN: 1932-6203
• Volume: 5
• Issue: 5
• Start Page: e10595
• End Page: e10595
• Grant/Funding Information: Development and maintenance of the PubMLST site used in this study has been funded by the Wellcome Trust.; This work was supported by a developmental grant from Emory University School of Medicine to TDR, grant TMTI0068_07_NM_T from the Joint Science and Technology Office for Chemical and Biological Defense (JSTO-CBD), Defense Threat Reduction Agency Initiative to TDR and internal funding from Network Biosystems Inc.
• Research Categories: Biology, Genetics; Biology, Virology

Abstract

Background Multiple locus sequence typing (MLST) has become a central genotyping strategy for analysis of bacterial populations. The scheme involves de novo sequencing of 6–8 housekeeping loci to assign unique sequence types. In this work we adapted MLST to a rapid microfluidics platform in order to enhance speed and reduce laboratory labor time. Methodology/Principal Findings Using two integrated microfluidic devices, DNA was purified from 100 Bacillus cereus soil isolates, used as a template for multiplex amplification of 7 loci and sequenced on forward and reverse strands. The time on instrument from loading genomic DNA to generation of electropherograms was only 1.5 hours. We obtained full-length sequence of all seven MLST alleles from 84 representing 46 different Sequence Types. At least one allele could be sequenced from a further 15 strains. The nucleotide diversity of B. cereus isolated in this study from one location in Rockville, Maryland (0.04 substitutions per site) was found to be as great as the global collection of isolates. Conclusions/Significance Biogeographical investigation of pathogens is only one of a panoply of possible applications of microfluidics based MLST; others include microbiologic forensics, biothreat identification, and rapid characterization of human clinical samples.